Integrating Siderophore Substructures in Thiol-Based Metallo-β-Lactamase Inhibitors

Metallo beta lactamases (MBLs) are among the most problematic resistance mechanisms of multidrug-resistant Gram-negative pathogens due to their broad substrate spectrum and lack of approved inhibitors. In this study, we propose the integration of catechol substructures into the design of thiol-based MBL inhibitors, aiming at mimicking bacterial siderophores for the active uptake by the iron acquisition system of bacteria. We synthesised two catechol-containing MBL inhibitors, as well as their dimethoxy counterparts, and tested them for in vitro inhibitory activity against NDM-1, VIM-1, and IMP-7. We demonstrated that the most potent catechol-containing MBL inhibitor is able to bind Fe3+ ions. Finally, we could show that this compound restores the antibiotic activity of imipenem in NDM-1-expressing K. pneumoniae, while leaving HUVEC cells completely unaffected. Thus, siderophore-containing MBL inhibitors might be a valuable strategy to overcome bacterial MBL-mediated resistance to beta lactam antibiotics.


Introduction
Multidrug-resistant Gram-negative pathogens are regarded as one of the major threats for human health. In the fight against these pathogens, approved anti-infectives are becoming increasingly ineffective. β-Lactamases, which inactivate β-lactam antibiotics, are produced by many multi-resistant Gram-negative bacteria and represent the most effective resistance mechanisms. Therefore, the inhibitors of β-lactamases are considered as effective antibiotic adjuvants, which can restore the activity of β-lactam antibiotics [1]. β-Lactamases can be separated in accordance to the underlying hydrolysis mechanism. Serine-β-lactamases (SBLs) use a nucleophilic serine to cleave the β-lactam core, while metallo-β-lactamases (MBLs) enable the nucleophilic attack of an activated water molecule, coordinated by one or two Zn 2+ ions in the active site [2]. While a number of SBL inhibitors, in combination with ß-lactam antibiotics, are approved for therapy, no inhibitor of MBLs has yet been approved. The most advanced MBL inhibitor, taniborbactam (Figure 1), is currently under investigation in clinical phase III trials for the treatment of complicated urinary tract infections [3].
Taniborbactam exhibits a cyclic boronate moiety that mimics the transition state of the β-lactam hydrolysis and coordinates to Zn 2+ ions in the active site of MBLs [4]. Furthermore, Brem et al. recently reported that indole-2-caboxylate-based compound 58 is a β-lactam mimetic, which displays excellent in vitro and in vivo adjuvant activity in combination with meropenem [5]. Another attractive option to inhibit MBL is offered by thiol-containing compounds [6,7]. The thiol moiety is a soft Lewis base which tightly coordinates to Zn 2+ One of the major drawbacks of the previously reported thiol-containing inhibitors is the comparably high lipophilicity, which is considered to be a limiting factor for the transport of the inhibitor through the outer membrane of Gram-negative pathogens into periplasm [10]. One of the strategies to overcome this drawback is the exploitation of the bacterial iron-acquisition system, also known as siderophore system. Siderophores are high-affinity iron chelators that are produced and excreted by bacteria and are actively reimported as siderophore-iron complexes [11]. Covalent conjugates of siderophores with antibiotics are also known as sideromycins; they mimic the siderophore-iron complexes to be actively imported by bacteria [12]. Sideromycins are not only synthetic products, as naturally occurring sideromycins are produced, e.g., by Streptomyces species [13]. Various combinations of siderophores and antibiotic agents have been reported so far, ultimately leading to the recent approval of cifederocol ( Figure 1). Cifederocol is a synthetic sideromycin that comprises a cephalosporin antibiotic linked to a catechol moiety, which is responsible for iron chelation. This combination makes cifederocol a highly effective antibiotic active against multi-resistant pathogens [14,15]. In this study we applied the sideromycin concept to thiol-based MBL inhibitors by integrating catechol moieties. One of the major drawbacks of the previously reported thiol-containing inhibitors is the comparably high lipophilicity, which is considered to be a limiting factor for the transport of the inhibitor through the outer membrane of Gram-negative pathogens into periplasm [10]. One of the strategies to overcome this drawback is the exploitation of the bacterial iron-acquisition system, also known as siderophore system. Siderophores are high-affinity iron chelators that are produced and excreted by bacteria and are actively re-imported as siderophore-iron complexes [11]. Covalent conjugates of siderophores with antibiotics are also known as sideromycins; they mimic the siderophore-iron complexes to be actively imported by bacteria [12]. Sideromycins are not only synthetic products, as naturally occurring sideromycins are produced, e.g., by Streptomyces species [13]. Various combinations of siderophores and antibiotic agents have been reported so far, ultimately leading to the recent approval of cifederocol ( Figure 1). Cifederocol is a synthetic sideromycin that comprises a cephalosporin antibiotic linked to a catechol moiety, which is responsible for iron chelation. This combination makes cifederocol a highly effective antibiotic active against multi-resistant pathogens [14,15]. In this study we applied the sideromycin concept to thiol-based MBL inhibitors by integrating catechol moieties.

Design of Siderophore-Containing MBL Inhibitors
In this study, we report the incorporation of catechol moieties into thiol-based MBL inhibitors. Ma et al. [16] and us [10] have previously reported on D-pipecolic acid-based MBL inhibitors. The X-ray structure of compound 1 ((2R)-1-[(2S)-2-methyl-3-sulfanyl-propanoyl] piperidine-2-carboxylic acid) in complex with NDM-1 has been published by Ma et al. and guided our design strategy to introduce an iron chelating moiety. Visual examination of the NDM-1 in complex with compound 1 (PDB code 6LJ0 [16]) revealed that while the thiol and the carboxylate moieties of compound 1 are tightly involved in directed interactions with the protein, the α-methyl moiety of the 3-mercapto-2-methylpropanoic acid part (yellow arrow), as well as the 4-position of the D-pipecolic acid part (blue arrow), are exposed to solvent (Figure 2A). This observation is in perfect agreement with the structure-activity relationship studies performed by Büttner et al. where they could show that substitutions in these positions are tolerated by NDM-1, VIM-1, and IMP-7 [10]. Therefore, we proposed that compounds 2 and 3, which exhibit a catechol moiety at the methyl position, could be potential siderophore-containing MBL inhibitors ( Figure 2B).

Design of Siderophore-Containing MBL Inhibitors
In this study, we report the incorporation of catechol moieties into thiol-based MBL inhibitors. Ma et al. [16] and us [10] have previously reported on D-pipecolic acid-based MBL inhibitors. The X-ray structure of compound 1 ((2R)-1-[(2S)-2-methyl-3-sulfanylpropanoyl]piperidine-2-carboxylic acid) in complex with NDM-1 has been published by Ma et al. and guided our design strategy to introduce an iron chelating moiety. Visual examination of the NDM-1 in complex with compound 1 (PDB code 6LJ0 [16]) revealed that while the thiol and the carboxylate moieties of compound 1 are tightly involved in directed interactions with the protein, the α-methyl moiety of the 3-mercapto-2methylpropanoic acid part (yellow arrow), as well as the 4-position of the D-pipecolic acid part (blue arrow), are exposed to solvent (Figure 2A). This observation is in perfect agreement with the structure-activity relationship studies performed by Büttner et al. where they could show that substitutions in these positions are tolerated by NDM-1, VIM-1, and IMP-7 [10]. Therefore, we proposed that compounds 2 and 3, which exhibit a catechol moiety at the methyl position, could be potential siderophore-containing MBL inhibitors ( Figure 2B).

Synthesis of Potential Siderophore-Containing MBL Inhibitors 2 and 3
For the synthetic implementation of compounds 2 and 3, we followed the synthetic route established by Büttner et al. [10] (Scheme 1). The preparation of compound 2 started from 2,3-dimethoxy benzaldehyde 4, which was coupled in a Knoevenagel-like condensation with Meldrum's acid to obtain 5. The reduction of 5 was accomplished with sodium borohydride. A Mannich-like cycloelimination of intermediate 6 with Eschenmoser´s salt led to methyl acrylate 7, which was subsequently converted to acrylic acid 8 via ester hydrolysis. Compound 8 was coupled with tBu-protected R-pipecolinic acid 19 in a Schotten-Baumann reaction. The Michael addition of thioacetic acid to the double bond of 9 yielded the fully protected precursor 10. Three deprotection steps led to the desired compound 2, wherein the dimethoxy intermediate 12 was also characterised as a final compound for use as potential non-chelating control.

Synthesis of Potential Siderophore-Containing MBL Inhibitors 2 and 3
For the synthetic implementation of compounds 2 and 3, we followed the synthetic route established by Büttner et al. [10] (Scheme 1). The preparation of compound 2 started from 2,3-dimethoxy benzaldehyde 4, which was coupled in a Knoevenagel-like condensation with Meldrum's acid to obtain 5. The reduction of 5 was accomplished with sodium borohydride. A Mannich-like cycloelimination of intermediate 6 with Eschenmoser s salt led to methyl acrylate 7, which was subsequently converted to acrylic acid 8 via ester hydrolysis. Compound 8 was coupled with tBu-protected R-pipecolinic acid 19 in a Schotten-Baumann reaction. The Michael addition of thioacetic acid to the double bond of 9 yielded the fully protected precursor 10. Three deprotection steps led to the desired compound 2, wherein the dimethoxy intermediate 12 was also characterised as a final compound for use as potential non-chelating control.
The second designed siderophore-containing MBL inhibitor 3 was obtained starting from 3,4-Dimethoxyphenylpropanoic acid 13, which was coupled with 1-Boc-protected Piperazine-2-carboxylic acid in a Schotten-Baumann reaction (Scheme 2). The free carboxylate 14 was protected by benzylation to obtain compound 15, which was subsequently deprotected at N1 by the cleavage of the Boc group under acidic conditions. Mercaptomethylpropionic acid was coupled to the hydrochloride 16, again under Schotten-Baumann conditions. Deprotection steps yielded the desired compound 3, while the dimethoxy precursor 18 was also characterised (vide supra). The second designed siderophore-containing MBL inhibitor 3 was obtained starting from 3,4-Dimethoxyphenylpropanoic acid 13, which was coupled with 1-Boc-protected Piperazine-2-carboxylic acid in a Schotten-Baumann reaction (Scheme 2). The free carboxylate 14 was protected by benzylation to obtain compound 15, which was subsequently deprotected at N1 by the cleavage of the Boc group under acidic conditions. Mercaptomethylpropionic acid was coupled to the hydrochloride 16, again under Schotten-Baumann conditions. Deprotection steps yielded the desired compound 3, while the dimethoxy precursor 18 was also characterised (vide supra).

Experimental Evaluation
The potential siderophore-containing MBL inhibitors 2 and 3, as well as their dimethoxy counterparts 12 and 18, were tested in a fluorescence-based enzyme activity assay. The MBLs VIM-1, IMP-7, and NDM-1 were recombinantly expressed in E. coli and the enzymatic activity was monitored by the conversion of the fluorogenic substrate The second designed siderophore-containing MBL inhibitor 3 was obtained starting from 3,4-Dimethoxyphenylpropanoic acid 13, which was coupled with 1-Boc-protected Piperazine-2-carboxylic acid in a Schotten-Baumann reaction (Scheme 2). The free carboxylate 14 was protected by benzylation to obtain compound 15, which was subsequently deprotected at N1 by the cleavage of the Boc group under acidic conditions. Mercaptomethylpropionic acid was coupled to the hydrochloride 16, again under Schotten-Baumann conditions. Deprotection steps yielded the desired compound 3, while the dimethoxy precursor 18 was also characterised (vide supra).

Experimental Evaluation
The potential siderophore-containing MBL inhibitors 2 and 3, as well as their dimethoxy counterparts 12 and 18, were tested in a fluorescence-based enzyme activity assay. The MBLs VIM-1, IMP-7, and NDM-1 were recombinantly expressed in E. coli and the enzymatic activity was monitored by the conversion of the fluorogenic substrate

Experimental Evaluation
The potential siderophore-containing MBL inhibitors 2 and 3, as well as their dimethoxy counterparts 12 and 18, were tested in a fluorescence-based enzyme activity assay. The MBLs VIM-1, IMP-7, and NDM-1 were recombinantly expressed in E. coli and the enzymatic activity was monitored by the conversion of the fluorogenic substrate fluorocillin [17], as described before [18,19]. All compounds were able to inhibit the selected MBLs in a low micromolar to submicromolar concentration range (Table 1). Interesting trends could be observed regarding compounds 2 and 3 and their dimethoxy counterparts 12 and 18. The free catechol moiety of compound 2 led to an improvement of inhibitory potency against VIM-1, while NDM-1 and IMP-7 tolerated both compounds, 2 and 12, equally well. In contrast, the deprotected catechol 3 outperformed the dimethoxy counterpart 18 in the inhibition of all three tested MBLs. Furthermore, in contrast to compound 2, the catechol inhibitor 3 exhibited submicromolar activity towards all three MBLs, which qualifies it for further evaluation. Complicated infections are often treated by the intravenous application of anti-infectives. Hence, the inner lining of the blood vessels, called endothelium, is highly prone to damages caused by the cytotoxic actions of these drugs. Therefore, we evaluated the potential cytotoxic effects of compounds 2, 3, 12, and 18 in vitro, using primary human umbilical vein endothelial cells (HUVECs). We found that concentrations of up to 128 µg/mL of compounds 2, 3, 12, and 18 neither influenced the cell viability ( Figure 3A) nor the apoptosis rate ( Figure 3B) of HUVECs in a negative manner. We investigated the potential of compound 3 to bind Fe 3+ ions via isothermal titrati calorimetry ( Figure 4). In this context, we titrated 250 µM of FeCl3 to 50 µM of compoun 3 and observed a binding event. When fitting the data with a model of independe binding, we observed that n = 0.467 ± 0.112, which approximately corresponds to t stoichiometry of 2:1 for the 3:Fe 3+ complex. These findings are consistent with t The considerable inhibition of purified MBLs in vitro, especially of NDM-1, with compound 3 suggested that these MBL inhibitors can potentially restore the activity of imipenem against MBL-expressing bacteria. To prove this, imipenem combined with different concentrations of mjr derivatives ranging from 2 to 128 mg/L was tested against a clinical K. pneumoniae isolate expressing NDM-1 ( Table 2). The most potent inhibitor, i.e., compound 3, significantly reduced the MIC of imipenem by 16-fold. We investigated the potential of compound 3 to bind Fe 3+ ions via isothermal titration calorimetry ( Figure 4). In this context, we titrated 250 µM of FeCl 3 to 50 µM of compound 3 and observed a binding event. When fitting the data with a model of independent binding, we observed that n = 0.467 ± 0.112, which approximately corresponds to the stoichiometry of 2:1 for the 3:Fe 3+ complex. These findings are consistent with the published data, e.g., 2:1 complexes of siderophores with iron are reported for the A. baumanii siderophore acinetobactin [20].  We examined the potential binding mode of compound 3 in complex with NDM-1. Therefore, we manually extended the C4-position of the D-pipecolic acid of compound 3 in complex with NDM-1 (PDB code 6LJ0) using the 3,4-dihydroxyphenyl propanoic acid residue, and subsequently performed an energy minimization of the complex ( Figure 5). We observed the ionic interactions of the carboxylate moiety bindings towards Lys211, while the catechol moiety extended into a more solvent-accessible binding site, where it formed water-mediated H bonds towards the backbone NH of His250. We examined the potential binding mode of compound 3 in complex with NDM-1. Therefore, we manually extended the C4-position of the D-pipecolic acid of compound 3 in complex with NDM-1 (PDB code 6LJ0) using the 3,4-dihydroxyphenyl propanoic acid residue, and subsequently performed an energy minimization of the complex ( Figure 5). We observed the ionic interactions of the carboxylate moiety bindings towards Lys211, while the catechol moiety extended into a more solvent-accessible binding site, where it formed water-mediated H bonds towards the backbone NH of His250.

Discussion
In this study we aimed for the design and synthesis of siderophore-containing MBL inhibitors. Compound 2 potently inhibited VIM-1 and IMP-7, however, it failed to inhibit NDM-1 in submicromolar range. Compound 3, which extends the D-pipecolic acid-based MBL inhibitors, inhibited all tested MBLs in submicromolar range. Thereby, it is more potent than captopril or tiopronin, which exhibited inhibitory values in a low micromolar range in the same activity assay performed in our group [8]. These findings suggest that future studies to design siderophore-containing MBL inhibitors should concentrate on the extension of the D-pipecolic acid in order to elaborate on the structure-activity relationships of this compound class. Furthermore, the efficacy of the siderophore should be improved. Different iron-chelating moieties, which had already served as useful siderophores in the past [21], could be coupled with D-pipecolic acid. Although the ironchelating affinity of compound 3 can be considered as mediocre, compound 3 exhibited a significant improvement in the MIC of imipenem in a clinical K. pneumoniae isolate, expressing NDM-1 at concentration of 16 µg/mL. Compared to compound 58 [5], the adjuvant activity of compound 3 can be further improved. Extensive further characterization of a range of different isolates should be performed in order to evaluate the potential of compound 3. Taken together, this study is the first report of an MBL inhibitor that contains a siderophore moiety which displays potent inhibitory activity against recombinant MBLs in vitro, and restores the antimicrobial activity of imipenem in a clinical K. pneumoniae isolate without affecting primary human cells. Figure 5. Proposed binding mode of compound 3 to NDM-1. Starting from the X-ray structure of compound 1 in complex with NDM-1 (PDB code 6LJ0), the catechol moiety was introduced manually and the complex was subsequently energy minimised.

Discussion
In this study we aimed for the design and synthesis of siderophore-containing MBL inhibitors. Compound 2 potently inhibited VIM-1 and IMP-7, however, it failed to inhibit NDM-1 in submicromolar range. Compound 3, which extends the D-pipecolic acid-based MBL inhibitors, inhibited all tested MBLs in submicromolar range. Thereby, it is more potent than captopril or tiopronin, which exhibited inhibitory values in a low micromolar range in the same activity assay performed in our group [8]. These findings suggest that future studies to design siderophore-containing MBL inhibitors should concentrate on the extension of the D-pipecolic acid in order to elaborate on the structure-activity relationships of this compound class. Furthermore, the efficacy of the siderophore should be improved. Different iron-chelating moieties, which had already served as useful siderophores in the past [21], could be coupled with D-pipecolic acid. Although the iron-chelating affinity of compound 3 can be considered as mediocre, compound 3 exhibited a significant improvement in the MIC of imipenem in a clinical K. pneumoniae isolate, expressing NDM-1 at concentration of 16 µg/mL. Compared to compound 58 [5], the adjuvant activity of compound 3 can be further improved. Extensive further characterization of a range of different isolates should be performed in order to evaluate the potential of compound 3. Taken together, this study is the first report of an MBL inhibitor that contains a siderophore moiety which displays potent inhibitory activity against recombinant MBLs in vitro, and restores the antimicrobial activity of imipenem in a clinical K. pneumoniae isolate without affecting primary human cells.

General Information
Reagents and solvents were purchased from the suppliers BLD Pharmtech GmbH (Kaiserslautern, Germany), Sigma Aldrich (Darmstadt, Germany), TCI Europe N.V. (Zwijndrecht, Belgium), or Fluorochem Ltd. (Derbyshire, United Kingdom), and used without further purification. Flash chromatography was performed on packed silica columns (particle size 50 µM) from Interchim (Montlucon Cedex, France) and with solvents at technical grade (for mixtures, see the corresponding experiments). Analytical tin layer chromatography (TLC) was performed with F254 TLC plates from Merck KGaA (Darmstadt, Germany); aromatic systems were visualised with ultraviolet light (240 nm). NMR spectra were measured on an AV 400, AV 500, or DRX 600 nuclear magnetic resonance spectrometer from Bruker (Karlsruhe, Germany). Chemical shifts were reported in parts per million (ppm), using TMS as an external standard, and the residual proton signal of the deuterated solvents (DMSO-d6) were used as an internal standard. The following abbreviations are used for the multiplicity of the signals: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and doublet of doublets (dd). The coupling constant (J) was determined by a machine and expressed in Hertz (Hz). HPLC and mass analysis were performed on a LCMS 2020 from Shimadzu (Duisburg, Germany). For analytical determination, a Luna 10u C18(2) (250 × 4.6 nm) was used, and for semi-preparative purification, a Luna 10µ C18(2) (250 × 21.20 nm) column from Phenomenex LTD Deutschland (Aschaffenburg, Germany) was used. The system is equipped with a SPD 20A UV/VIS detector (λ = 240/280 nm) and an ESI-TOF (measuring in the positive-and/or negative-ion mode). Eluent mixtures of acetonitrile/0.1% aqueous formic acid were used, with a flow rate of 0.1 mL/min (Scout Column) or 21 mL/min (semi-preparative) at room temperature. The following methods were used: method 1 was 0 min 95% ACN, 2 min 95% ACN, 14 min 10% ACN, and 20 min 10% ACN; and method 2 was 0 min 50% ACN, 10 min 10% ACN, and 15 min 10% ACN. The purity of the compounds was determined by integrating the peaks of the UV chromatogram. All compounds were found to be >95% pure through HPLC analysis. Further MS spectra were obtained on a Thermo Fisher Surveyor MSQ coupled with a Camag TLC-MS interface 2. High-resolution mass spectra were recorded on an MALDI LTQ ORBITRAP XL instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA). The matrix used was α-cyano-4-hydroxycinnamic acid (HCCA). The structure of all compounds presented were verified by a least two of the described methods, and the final compounds were verified by NMR, Mass, HRMS and HPLC to determine a purity of >95%.

Enzymatic Activity Assays
The enzymatic activity assays relied on the cleavage of the fluorogenic substrate fluorocillin with the recombinant MBLs [8]. Fluorocillin was synthetised, as described by Rukavishnikov et al. [17]. The MBLs VIM-1, IMP-7, and NDM-1 were recombinantly expressed in E. coli and were diluted in an assay buffer (HEPES 50 mM, pH 7.5; 0.01 %Triton X-100). The final protein concentration of the MBLs and supplemented ZnCl 2 was: VIM-1-4 nM, IMP-7-0.1 nM, and NDM-1-3 nM. 1 µL of the inhibitors dissolved in DMSO was added to 89 µL of the protein solution. After 30 min of incubation with the inhibitors, 10 µL of fluorocillin solution in an assay buffer (888 nM fluorocillin, HEPES 50 mM, pH 7.5; 0.01 %Triton X-100) was added (100 µL final assay volume; 1% final DMSO concentration) and the fluorescence gain was measured for 30 cycles using a Tecan fluorescent plate reader (Infinite F200; excitation at 495 nm and emission at 525 nm). Negative controls were measured in the absence of enzymes (89 µL of assay buffer), whereas the positive controls were measured in the presence of enzymes and in the absence of inhibitors (1 µL of DMSO). The inhibitory effect of each substance was measured in triplicate in three independent experiments. IC 50 values were calculated using the data obtained from measurements with at least eight different inhibitor concentrations, applying a sigmoidal dose-response (variable slope with four

Molecular Modelling Studies
The X-ray structure of compound 1 in complex with NDM-1 (PDB code 6LJ0; [16]), was prepared for molecular modelling using the QuickPrep routine of the MOE2019.0102 software (Chemical Computing Group; Montreal, QC, Canada), which included the modelling of unresolved loops and the adjustment of the protonation state, as well as energy minimization. The catechol moiety was introduced manually using the builder tool, and the complex was subsequently energy minimised.